At serotonergic synapse, serotonin (5-HT) transporters (SERTs) dictate the concentration and duration of synaptic 5-HT by re-uptake of released 5-HT into the presynaptic terminals. Therefore, the activity of SERT in the presynaptic plasma membrane is a critical factor that determines the strength of serotonergic neurotransmission and hence behavioral and physiological functions. SERTs are high-affinity molecular targets for several addictive and therapeutic drugs including cocaine, amphetamine, MDMA "ecstasy", tricyclic antidepressants and SSRIs. In our previous funding period, we, and collaborators identified the signaling mechanisms of acute SERT regulation by several protein kinases, and documented lipid raft-mediated redistribution of amine transporters in native and in vitro model systems. However, the physiological relevance of SERT phosphorylation in 5-HT transport regulation, and the sites or domains responsible have yet to be examined. The major goal of this renewal application is to determine the physiological role of SERT phosphorylation in normal 5-HT transport regulation and in dysregulations associated with human SERT coding variants of obsessive-compulsive disorder (OCD) and other disease phenotypes. Specific Aim 1 will test the hypothesis that protein kinases PKG and p38 MARK phosphorylate SERT on specific phospho-sites/motifs following histamine receptor (HSR) activation and regulate 5HT transport. Specific Aim 2 will examine whether hSERT coding variants exist in altered phosphorylation state and are insensitive to H3R/PKG/p38 MARK modulation. Specific Aim 3 will test the hypotheses that in vivo modulation of HSR alters SERT phosphorylation regulating 5HT transport and hence synaptic 5HT levels. The studies proposed in this application will (i) determine the physiologically relevant signals/receptors influencing 5-HT transport;(ii) define the mechanistic link between transporter phosphorylation and 5-HT transport;(iii) establish the cellular machinery responsible for SERT distribution;and (iv) provide information on dysregulations associated with hSERT variants that could be of use in the development of new therapeutic strategies aimed at regulating SERT function in the treatment of mental illnesses such as OCD, depression and drug abuse.